Targeted genome editing across species using ZFNs and TALENs

Abstract

Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species.

Fig. 1

Heritable, targeted nematode mutations induced by ZFNs and TALENs. (A) Hermaphrodite gonads with an intact (left) or ZFN-disrupted (right) GFP::histone transgene. Residual signal (right) is gut autofluorescence. (B) Frequency of ben-1 mutants (left y axis, red line) from time periods after ZFN mRNA injection (x axis) relative to total progeny per period (right y axis, histogram). (C) ZFN-induced mutations in cis regulatory element rex-1 cause partial deletion and full duplication (asterisks) of MEX motif (yellow). Represented are ZFN recognition sequences (underlined), FokI cleavage region (lowercase nucleotides), insertions (green), and deletions (red colons). (D) TALEN recognition sequences in ben-1 and TALEN-induced mutation. Each RVD (colored blocks) recognizes a nucleotide (NT) in the target site. (E) Proportion of each mutation class induced by ZFNs (n=102) and TALENs (n = 27).